Induction of myelinating oligodendrocytes in human cortical spheroids.

Cerebral organoids provide an accessible system for investigations of cellular composition, interactions, and organization but have lacked oligodendrocytes, the myelinating glia of the central nervous system. Here we reproducibly generated oligodendrocytes and myelin in 'oligocortical spheroids' derived from human pluripotent stem cells. Molecular features consistent with those of maturing oligodendrocytes and early myelin appeared by week 20 in culture, with further maturation and myelin compaction evident by week 30. Promyelinating drugs enhanced the rate and extent of oligodendrocyte generation and myelination, and spheroids generated from human subjects with a genetic myelin disorder recapitulated human disease phenotypes. Oligocortical spheroids provide a versatile platform for studies of myelination of the developing central nervous system and offer new opportunities for disease modeling and therapeutic development.

Massive pulmonary embolism in patients with extreme bleeding risk: a case series on the successful use of ultrasound-assisted, catheter directed thrombolysis in a district general hospital.

Massive pulmonary embolism (PE), characterised by profound arterial hypotension, is a life-threatening emergency with a 90-day mortality of over 50%. Systemic thrombolysis can significantly reduce the risk of death or cardiovascular collapse in these patients, by around 50%, but these benefits are offset by a fivefold increased risk of intracranial haemorrhage and major bleeding, which may limit its use in patients at high risk of catastrophic haemorrhage. We describe a case series of 3 patients presenting with massive PE, each with extreme risk of bleeding and contra-indication to systemic thrombolysis, treated successfully with ultrasound-assisted, catheter directed thrombolysis (U-ACDT). Our experience of this novel technique using the EkoSonic Endovascular System (Ekos, BTG, London, UK) on carefully selected patients has demonstrated the potential to improve clinical status in shocked patients, with minimal bleed risk. There have been several clinical studies evaluating the Ekos system. Both the ULTIMA and SEATTLE II studies have shown significant reductions in RV/LV ratio by CT scanning when compared to standard anticoagulation in patients with intermediate-risk PE, with minimal bleeding complications. However, there is a pressing need for a randomised trial demonstrating improvement in robust clinical outcomes when comparing U-ACDT to simple anticoagulation. We believe that this case series adds new insight and highlights the potential of catheter directed thrombolysis in this high-risk patient cohort and consideration should be made to its use in cases where systemic thrombolysis is felt to be too high risk.

The integrated stress response in hypoxia-induced diffuse white matter injury.

Currently no treatments exist for preterm infants with diffuse white matter injury (DWMI) caused by hypoxia. Due to improved care of preterm neonates and increased recognition by advanced imaging techniques, the prevalence of DWMI is increasing. A better understanding of the pathophysiology of DWMI is therefore of critical importance. The integrated stress response (ISR), a conserved eukaryotic response to myriad stressors including hypoxia, may play a role in hypoxia-induced DWMI and may represent a novel target for much needed therapies. In this study we utilize in vitro and in vivo hypoxic models of DWMI to investigate whether the ISR is involved in DWMI. We demonstrate that hypoxia activates the ISR in primary mouse oligodendrocyte precursor cells (OPCs) in vitro and that genetically inhibiting the ISR in differentiating OPCs increases their susceptibility to in vitro hypoxia. We also show that a well-established in vivo mild chronic hypoxia (MCH) mouse model and a new severe acute hypoxia (SAH) mouse model of DWMI activates the initial step of the ISR. Nonetheless, genetic inhibition of the ISR has no detectable effect on either MCH or SAH-induced DWMI. In addition, we demonstrate that genetic enhancement of the ISR does not ameliorate MCH or SAH-induced DWMI. These studies suggest that while the ISR protects OPCs from hypoxia in vitro, it does not appear to play a major role in either MCH or SAH-induced DWMI and is therefore not a likely target for therapies aimed at improving neurological outcome in preterm neonates with hypoxia-induced DWMI.SIGNIFICANCE STATEMENTDiffuse white matter injury (DWMI) caused by hypoxia is a leading cause of neurological deficits following premature birth. An increased understanding of the pathogenesis of this disease is critical. The integrated stress response (ISR) is activated by hypoxia and protects oligodendrocyte lineage cells in other disease models. This has led to an interest in the potential role of the ISR in DWMI. Here we examine the ISR in hypoxia-induced DWMI and show that while the ISR protects oligodendrocyte lineage cells from hypoxia in vitro, genetic inhibition or enhancement of the ISR has no effect on hypoxia-induced DWMI in vivo suggesting that the ISR does not play a major role in, and is not a likely therapeutic target for, DWMI.

Neonatal Hypoxia Results in Peripheral Nerve Abnormalities.

Although the adverse effects of neonatal hypoxia associated with premature birth on the central nervous system are well known, the contribution of hypoxic damage to the peripheral nervous system (PNS) has not been addressed. We demonstrate that neonatal hypoxia results in hypomyelination and delayed axonal sorting in mice leading to electrophysiological and motor deficits that persist into adulthood. These findings support a potential role for PNS hypoxic damage in the motor impairment that results from premature birth and suggest that therapies designed to protect the PNS may provide clinical benefit.

Safety of intravenous ß-adrenoceptor blockers for computed tomographic coronary angiography.

AIMS: To assess the safety of our clinical practice using off-label intravenous metoprolol to facilitate computed tomographic (CT) coronary angiography. METHODS: A retrospective analysis of scan reports and hospital admissions data was conducted to identify adverse events occurring following CT coronary angiography in patients who had received intravenous metoprolol prior to the scan. RESULTS: A total of 3098 patients were included, of whom 1871 received intravenous metoprolol. Nine hundred and one patients received more than 15 mg and 129 patients received metoprolol despite a resting heart rate <65 beats min(-1) . There was a single adverse incident, comprising transient loss of consciousness. CONCLUSIONS: The use of intravenous metoprolol to facilitate cardiac CT scanning appears safe. Dose limits recommended for other indications, generally in acutely unwell patients, may not need to be as stringent in this population.

Oligodendrocyte-specific activation of PERK signaling protects mice against experimental autoimmune encephalomyelitis.

There is compelling evidence that oligodendrocyte apoptosis, in response to CNS inflammation, contributes significantly to the development of the demyelinating disorder multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). Therefore, approaches designed to protect oligodendrocytes would likely have therapeutic value. Activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum (ER) stress increases cell survival under various cytotoxic conditions. Moreover, there is evidence that PERK signaling is activated in oligodendrocytes within demyelinating lesions in multiple sclerosis and EAE. Our previous study demonstrated that CNS delivery of the inflammatory cytokine interferon-γ before EAE onset protected mice against EAE, and this protection was dependent on PERK signaling. In our current study, we sought to elucidate the role of PERK signaling in oligodendrocytes during EAE. We generated transgenic mice that allow for temporally controlled activation of PERK signaling, in the absence of ER stress, specifically in oligodendrocytes. We demonstrated that persistent activation of PERK signaling was not deleterious to oligodendrocyte viability or the myelin of adult animals. Importantly, we found that enhanced activation of PERK signaling specifically in oligodendrocytes significantly attenuated EAE disease severity, which was associated with reduced oligodendrocyte apoptosis, demyelination, and axonal degeneration. This effect was not the result of an altered degree of the inflammatory response in EAE mice. Our results provide direct evidence that activation of PERK signaling in oligodendrocytes is cytoprotective, protecting mice against EAE.

Primary percutaneous coronary intervention for refractory cardiac arrest.

The identification and treatment of reversible causes is paramount to the success of resuscitation in cardiac arrest, particularly when standard therapy has failed. Acute coronary occlusion is one such cause, and the introduction of primary percutaneous coronary intervention services may provide an opportunity for emergency revascularization in this setting. This article describes 2 patients with cardiac arrest as a result of coronary occlusion, in which standard therapeutic measures proved futile. The first patient had refractory ventricular fibrillation, and the second had an episode of ventricular fibrillation followed by true pulseless electrical activity: total cessation of ventricular activity. In both examples, external mechanical compression and primary percutaneous coronary intervention facilitated coronary revascularization and achieved return of spontaneous circulation, leading to survival to hospital discharge.

Pervasive environmental chemicals impair oligodendrocyte development.

Exposure to environmental chemicals can impair neurodevelopment, and oligodendrocytes may be particularly vulnerable, as their development extends from gestation into adulthood. However, few environmental chemicals have been assessed for potential risks to oligodendrocytes. Here, using a high-throughput developmental screen in cultured cells, we identified environmental chemicals in two classes that disrupt oligodendrocyte development through distinct mechanisms. Quaternary compounds, ubiquitous in disinfecting agents and personal care products, were potently and selectively cytotoxic to developing oligodendrocytes, whereas organophosphate flame retardants, commonly found in household items such as furniture and electronics, prematurely arrested oligodendrocyte maturation. Chemicals from each class impaired oligodendrocyte development postnatally in mice and in a human 3D organoid model of prenatal cortical development. Analysis of epidemiological data showed that adverse neurodevelopmental outcomes were associated with childhood exposure to the top organophosphate flame retardant identified by our screen. This work identifies toxicological vulnerabilities for oligodendrocyte development and highlights the need for deeper scrutiny of these compounds' impacts on human health.

A phenotypic screening platform for identifying chemical modulators of astrocyte reactivity.

Disease, injury and aging induce pathological reactive astrocyte states that contribute to neurodegeneration. Modulating reactive astrocytes therefore represent an attractive therapeutic strategy. Here we describe the development of an astrocyte phenotypic screening platform for identifying chemical modulators of astrocyte reactivity. Leveraging this platform for chemical screening, we identify histone deacetylase 3 (HDAC3) inhibitors as effective suppressors of pathological astrocyte reactivity. We demonstrate that HDAC3 inhibition reduces molecular and functional characteristics of reactive astrocytes in vitro. Transcriptional and chromatin mapping studies show that HDAC3 inhibition disarms pathological astrocyte gene expression and function while promoting the expression of genes associated with beneficial astrocytes. Administration of RGFP966, a small molecule HDAC3 inhibitor, blocks reactive astrocyte formation and promotes neuroprotection in vivo in mice. Collectively, these results establish a platform for discovering modulators of reactive astrocyte states, inform the mechanisms that control astrocyte reactivity and demonstrate the therapeutic benefits of modulating astrocyte reactivity for neurodegenerative diseases.

Structural Characterization of an LPA1 Second Extracellular Loop Mimetic with a Self-Assembling Coiled-Coil Folding Constraint.

G protein-coupled receptor (GPCR) structures are of interest as a means to understand biological signal transduction and as tools for therapeutic discovery. The growing number of GPCR crystal structures demonstrates that the extracellular loops (EL) connecting the membrane-spanning helices show tremendous structural variability relative to the more structurally-conserved seven transmembrane α-helical domains. The EL of the LPA(1) receptor have not yet been conclusively resolved, and bear limited sequence identity to known structures. This study involved development of a peptide to characterize the intrinsic structure of the LPA(1) GPCR second EL. The loop was embedded between two helices that assemble into a coiled-coil, which served as a receptor-mimetic folding constraint (LPA(1)-CC-EL2 peptide). The ensemble of structures from multi-dimensional NMR experiments demonstrated that a robust coiled-coil formed without noticeable deformation due to the EL2 sequence. In contrast, the EL2 sequence showed well-defined structure only near its C-terminal residues. The NMR ensemble was combined with a computational model of the LPA(1) receptor that had previously been validated. The resulting hybrid models were evaluated using docking. Nine different hybrid models interacted with LPA 18:1 as expected, based on prior mutagenesis studies, and one was additionally consistent with antagonist affinity trends.

Pervasive environmental chemicals impair oligodendrocyte development.

Exposure to environmental chemicals can impair neurodevelopment1-4. Oligodendrocytes that wrap around axons to boost neurotransmission may be particularly vulnerable to chemical toxicity as they develop throughout fetal development and into adulthood5,6. However, few environmental chemicals have been assessed for potential risks to oligodendrocyte development. Here, we utilized a high-throughput developmental screen and human cortical brain organoids, which revealed environmental chemicals in two classes that disrupt oligodendrocyte development through distinct mechanisms. Quaternary compounds, ubiquitous in disinfecting agents, hair conditioners, and fabric softeners, were potently and selectively cytotoxic to developing oligodendrocytes through activation of the integrated stress response. Organophosphate flame retardants, commonly found in household items such as furniture and electronics, were non-cytotoxic but prematurely arrested oligodendrocyte maturation. Chemicals from each class impaired human oligodendrocyte development in a 3D organoid model of prenatal cortical development. In analysis of epidemiological data from the CDC's National Health and Nutrition Examination Survey, adverse neurodevelopmental outcomes were associated with childhood exposure to the top organophosphate flame retardant identified by our oligodendrocyte toxicity platform. Collectively, our work identifies toxicological vulnerabilities specific to oligodendrocyte development and highlights common household chemicals with high exposure risk to children that warrant deeper scrutiny for their impact on human health.

Patient iPSC models reveal glia-intrinsic phenotypes in multiple sclerosis.

Multiple sclerosis (MS) is considered an inflammatory and neurodegenerative disease of the central nervous system, typically resulting in significant neurological disability that worsens over time. While considerable progress has been made in defining the immune system's role in MS pathophysiology, the contribution of intrinsic CNS-cell dysfunction remains unclear. Here, we generated the largest reported collection of iPSC lines from people with MS spanning diverse clinical subtypes and differentiated them into glia-enriched cultures. Using single-cell transcriptomic profiling, we observed several distinguishing characteristics of MS cultures pointing to glia-intrinsic disease mechanisms. We found that iPSC-derived cultures from people with primary progressive MS contained fewer oligodendrocytes. Moreover, iPSC-oligodendrocyte lineage cells and astrocytes from people with MS showed increased expression of immune and inflammatory genes that match those of glial cells from MS postmortem brains. Thus, iPSC-derived MS models provide a unique platform for dissecting glial contributions to disease phenotypes independent of the peripheral immune system and identify potential glia-specific targets for therapeutic intervention.

Speg interactions that regulate the stability of excitation-contraction coupling protein complexes in triads and dyads.

Here we show that striated muscle preferentially expressed protein kinase α (Spegα) maintains cardiac function in hearts with Spegß deficiency. Speg is required for stability of excitation-contraction coupling (ECC) complexes and interacts with esterase D (Esd), Cardiomyopathy-Associated Protein 5 (Cmya5), and Fibronectin Type III and SPRY Domain Containing 2 (Fsd2) in cardiac and skeletal muscle. Mice with a sequence encoding a V5/HA tag inserted into the first exon of the Speg gene (HA-Speg mice) display a >90% decrease in Spegß but Spegα is expressed at ~50% of normal levels. Mice deficient in both Spegα and Speg ß (Speg KO mice) develop a severe dilated cardiomyopathy and muscle weakness and atrophy, but HA-Speg mice display mild muscle weakness with no cardiac involvement. Spegα in HA-Speg mice suppresses Ca2+ leak, proteolytic cleavage of Jph2, and disruption of transverse tubules. Despite it's low levels, HA-Spegß immunoprecipitation identified Esd, Cmya5 and Fsd2 as Spegß binding partners that localize to triads and dyads to stabilize ECC complexes. This study suggests that Spegα and Spegß display functional redundancy, identifies Esd, Cmya5 and Fsd2 as components of both cardiac dyads and skeletal muscle triads and lays the groundwork for the identification of new therapeutic targets for centronuclear myopathy.

Oligodendrocyte progenitor cell fate and function in development and disease.

Differentiation of oligodendrocyte progenitor cells (OPCs) into myelination-capable mature oligodendrocytes is essential for proper function of the central nervous system. OPCs are tissue-resident stem cells that populate all regions of the central nervous system and exist beyond development into adulthood. Disorders that lead to disruption of this critical cell state change cause devastating myelin diseases that are often associated with shortened life span. Recent findings have also provided support for a newly appreciated contribution of perturbed OPC differentiation to neurodegenerative and psychiatric diseases. These findings emphasize the need for a more complete understanding of OPC differentiation in health and disease. Here, we review recent molecular and functional findings revealing new roles of OPCs. It is our hope that this review provides readers with an enticing snapshot of current OPC research and highlights the potential of controlling OPC fate and function to treat diseases of the brain.

Non-canonical Targets of HIF1a Impair Oligodendrocyte Progenitor Cell Function.

Mammalian cells respond to insufficient oxygen through transcriptional regulators called hypoxia-inducible factors (HIFs). Although transiently protective, prolonged HIF activity drives distinct pathological responses in different tissues. Using a model of chronic HIF1a accumulation in pluripotent-stem-cell-derived oligodendrocyte progenitors (OPCs), we demonstrate that HIF1a activates non-canonical targets to impair generation of oligodendrocytes from OPCs. HIF1a activated a unique set of genes in OPCs through interaction with the OPC-specific transcription factor OLIG2. Non-canonical targets, including Ascl2 and Dlx3, were sufficient to block differentiation through suppression of the oligodendrocyte regulator Sox10. Chemical screening revealed that inhibition of MEK/ERK signaling overcame the HIF1a-mediated block in oligodendrocyte generation by restoring Sox10 expression without affecting canonical HIF1a activity. MEK/ERK inhibition also drove oligodendrocyte formation in hypoxic regions of human oligocortical spheroids. This work defines mechanisms by which HIF1a impairs oligodendrocyte formation and establishes that cell-type-specific HIF1a targets perturb cell function in response to low oxygen.

Developing therapeutic strategies to promote myelin repair in multiple sclerosis.

Introduction: Approved disease-modifying therapies for multiple sclerosis (MS) lessen inflammatory disease activity that causes relapses and MRI lesions. However, chronic inflammation and demyelination lead to axonal degeneration and neuronal loss, for which there currently is no effective treatment. There has been increasing interest in developing repair-promoting strategies, but there are important unanswered questions regarding the mechanisms and appropriate methods to evaluate these treatments. Areas covered: The rationale for remyelinating agents in MS is discussed, with an overview of both myelin physiology and endogenous repair mechanisms. This is followed by a discussion of the identification and development of potential remyelinating drugs. Potential biomarkers of remyelination are reviewed, including considerations regarding measuring remyelination in clinical trials. Information and data were obtained from a search of recent literature through PubMed. Peer-reviewed original articles and review articles were included. Expert opinion: There are several obstacles to the translation of potential remyelinating agents to clinical trials, particularly uncertainty regarding the most appropriate study population and method to monitor remyelination. Refinements in clinical trial design and outcome measurement, potentially via advanced imaging techniques, are needed to optimize detection of repair in patients with MS.

Risk of thromboembolism in patients developing critical illness-associated atrial fibrillation.

Although common, the long-term significance of -developing atrial fibrillation (AF) during a period of critical illness is unclear. We undertook a retrospective cohort analysis to -assess the rate of thromboembolism (TE) in patients -developing atrial fibrillation de novo during admission to our intensive care unit. In total, 1,955 patients were followed up (-maximum follow-up 1,276 days) for the occurrence of TE, of which 220 (11.3%) had developed AF or atrial flutter during their critical care admission. There were 11 TE events among the patients with new AF (0.053 events per patient-year), compared with 18 in the non-AF group (0.0059 events per patient-year). The unadjusted hazard ratio for TE in patients developing new AF compared with those not developing AF was 8.09 (95% CI 3.08-17.19, p<0.001). In patients admitted to critical care, the development of AF appears to be associated with a significantly increased risk of subsequent thromboembolism.

Rapid functional genetics of the oligodendrocyte lineage using pluripotent stem cells.

Oligodendrocyte dysfunction underlies many neurological disorders, but rapid assessment of mutation-specific effects in these cells has been impractical. To enable functional genetics in oligodendrocytes, here we report a highly efficient method for generating oligodendrocytes and their progenitors from mouse embryonic and induced pluripotent stem cells, independent of mouse strain or mutational status. We demonstrate that this approach, when combined with genome engineering, provides a powerful platform for the expeditious study of genotype-phenotype relationships in oligodendrocytes.

Chemical Screening Identifies Enhancers of Mutant Oligodendrocyte Survival and Unmasks a Distinct Pathological Phase in Pelizaeus-Merzbacher Disease.

Pelizaeus-Merzbacher disease (PMD) is a fatal X-linked disorder caused by loss of myelinating oligodendrocytes and consequent hypomyelination. The underlying cellular and molecular dysfunctions are not fully defined, but therapeutic enhancement of oligodendrocyte survival could restore functional myelination in patients. Here we generated pure, scalable quantities of induced pluripotent stem cell-derived oligodendrocyte progenitor cells (OPCs) from a severe mouse model of PMD, Plp1jimpy. Temporal phenotypic and transcriptomic studies defined an early pathological window characterized by endoplasmic reticulum (ER) stress and cell death as OPCs exit their progenitor state. High-throughput phenotypic screening identified a compound, Ro 25-6981, which modulates the ER stress response and rescues mutant oligodendrocyte survival in jimpy, in vitro and in vivo, and in human PMD oligocortical spheroids. Surprisingly, increasing oligodendrocyte survival did not restore subsequent myelination, revealing a second pathological phase. Collectively, our work shows that PMD oligodendrocyte loss can be rescued pharmacologically and defines a need for multifactorial intervention to restore myelination.